Atorvastatin is well known as an anti-hyperlipidemic agent, an antihypercholesterolemic agent, or a cholesterol-lowering agent. Oxidative metabolism of atorvastatin in human liver is mediated by mainly cytochrome P450 3A (CYP3A) enzymes, particularly, cytochrome P450 3A4 (CYP3A4), and the following two metabolites, that is, ortho-hydroxy atorvastatin (ortho-OH atorvastatin or 2-OH atorvastatin) and parahydroxy atorvastatin (para-OH atorvastatin or 4-OH atorvastatin) are generated.

After oral ingestion, atorvastatin, which is an inactive lactone, is hydrolyzed to the corresponding β-hydroxy acid form. This is a main metabolite and an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. HMG-CoA reductase catalyzes the conversion of HMG-CoA to mevalonate, which is an early and rate-limiting step in the biosynthesis of cholesterol.
In addition to the P450-mediated oxidation and β-oxidation processes, glucuronidation constitutes a common metabolic pathway for statins (Prueksaritanont et al., Drug Metab. Dispos. 30:505-512, 2002). The metabolites resulting from microsomal oxidation of atorvastatin by P450 enzymes are effective inhibitors of HMG-CoA reductase. In addition, it has been suggested that the metabolites may contribute to the cholesterol-lowering effect of atorvastatin.
Cytochrome P450 enzymes (P450s or CYPs) are large families consisting of enzymes serving as remarkably diverse oxygenation catalysts in throughout nature from archaea, bacteria, fungi, plants, and animals up to humans (http://drnelson.uthsc.edu/CytochromeP450.html). Due to the catalytic diversity and broad substrate range of P450s, they are attractive biocatalyst candidates for the production of fine chemicals, including pharmaceuticals.
However, in spite of the potential use of mammalian P450s in various biotechnology fields, they are not suitable as biocatalysts because of their low stability, low catalytic activity, and low affordability.
In the case in which a pro-drug is converted into a biologically “active metabolite” by human hepatic P450s during drug development, a large amount of pure metabolites are required in order to research into effect, toxicity, pharmacokinetics of the drug, or the like. Further, in the case in which the metabolite itself has biological activity, it may be advantageous to directly administer the metabolite to the body. Therefore, it is important to prepare the metabolite on a large scale.
However, since there are various problems in chemically synthesizing pure metabolites, P450 may be used in order to prepare the metabolites of a drug or drug candidates as an alternative for chemical synthesis of the metabolites. The metabolite preparation has been reported using human P450s expressed in Escherichia coli (Yun et al., Curr. Drug Metab. 7:411-429, 2006) and in insect cells (Rushmore et al., Metab. Eng. 2:115-125, 2000; Vail et al., J. Ind. Microbiol. Biotechnol. 32:67-74. 2005).
However, since these systems are still costly and have low productivities due to limited stabilities and slow reaction rates, a method of using engineered bacterial P450 enzymes having the desired catalyst activity has been suggested as an alternative for producing human metabolite.
Meanwhile, P450 BM3 (CYP102A1) from Bacillus megaterium has strong similarity to eukaryotic members of the CYP4A (fatty acid hydroxylase) family. It has been reported that CYP102A1 mutants oxidizes several human P450 substrates to produce the metabolite with higher activity (Kim et al., Protein Expr. Purif. 57:188-200, 2008a). Further, CYP102A1 is a versatile monooxygenase capable of working on various substrates (Di Nardo et al., J. Biol. Inorg. Chem. 12:313-323, 2007).
Recently, it has been reported that CYP102A1 mutants may produce larger quantities of the human metabolites of drugs, which may be difficult to be synthesized (Otey et al., Biotechnol. Bioeng. 93:494-499, 2005). Therefore, as an alternative method of preparing the metabolites, it may be considered to use CYP102A1 engineered so as to have the desired properties.
Several amino acid residues in CYP102A1 were mutated to generate mutant enzymes having increased activity toward human P450 substrates by the present inventors (Yun et al., Trends Biotechnol. 25:289-298, 2007 and other references cited in the article), and it was confirmed that specific mutants among these mutant enzymes may enable the CYP102A1 enzyme to catalyze O-deethylation and 3-hydroxylation of 7-ethoxycoumarin (Kim et al. Drug Metab. Dispos. 36:2166-2170, 2008a).
Therefore, while conducting research for directly using the atorvastatin metabolites as a drug, the present inventors discovered bacterial enzymes capable of oxidizing atorvastatin, which is known as a human P450 substrate, to produce 2-hydroxylated product and 4-hydroxylated product, which are human metabolites, and a biological preparation method using the same, thereby completing the present invention.